Name: assessing stem cell dna integrity for cardiac cell therapy
Uploaded: 2019-01-25T14:00:00.000+00:00
Duration: 10 min 16 s
Description: Watch this Scientific Journal Video about Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy at JoVE.com

This work was supported in part by National Heart, Lung, and Blood Institute Grants RO1-HL-99507, HL-114120, HL-131017, HL138023, and UO1-HL-134764 (to J. Zhang) and by American Heart Association Scientific Development Grant 17SDG33670677 (to R. Kannappan).

1. Comet Assay
<ol>
	<li>Reagent preparation
	<ol>
		<li>For low-melting-point agarose, place 500 mg of low-melting agarose in 100 mL of DNA-/RNA-free water. Heat the bottle in a microwave oven until the agarose dissolves. Place the bottle in a 37 &#176;C water bath until needed. 
		NOTE: For further reading, see Azqueta et al., who studied the effects of agarose concentration on the DNA-unwinding time and several electrophoresis factors23.</li>
		<li>Dilute SYBR green DNA dye in Tris-EDTA (TE) Buffer at a 1:10,000 ratio to obtain a 1x working stock dye solution. This dye is light-sensitive, so store it in a dark place.</li>
		<li>For the cell lysis buffer, dissolve 100 mL of lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 1% Triton X-100) in deionized water (DI H2O). Then, add 10 M NaOH to adjust the pH to 10.0. Cool the buffer to 4 &#176;C.</li>
		<li>For the alkaline DNA-unwinding/electrophoresis running buffer, dissolve 1 L of alkaline solution (0.3 M NaOH, 1 mM EDTA) in DI H2O. Cool the buffer to 4 &#176;C. Make sure the pH &#62;13.</li>
		<li>For precoated comet slides, place a few drops of 0.5% agarose on a glass slide. Immediately, using the flat surface of another slide, spread the agarose to form a thin layer of agarose coating. 
		NOTE: Dry the slides before using.</li>
		<li>For iPS cell culture, briefly culture iPS cells in basement-membrane-matrix-coated (see Table of Materials) culture plates in mTESR1 culture medium until confluency is reached. 
		NOTE: Human-cardiac-fibroblast-derived iPS cells were used in this protocol. The reprogramming of iPS cell derivation and culture conditions is described in recent articles from our research group12,13,21.</li>
	</ol>
	</li>
	<li>Sample preparation
	<ol>
		<li>Discard the culture medium and wash the cells. Dissociate the cells using detachment solution (see Table of Materials). Wash the cell pellet with phosphate-buffered saline (PBS) and resuspend the cells in PBS at 1 x 105 cells/mL.</li>
	</ol>
	</li>
	<li>Sample slide preparation 
	NOTE: Perform the following steps under low-light conditions to avoid ultraviolet-light-induced cell damage.
	<ol>
		<li>Mix 10 &#956;L of cell suspension and 90 &#956;L of agarose solution (1:10 ratio) in a tube. Place the tube in a 37 &#176;C water bath to prevent the agarose from solidifying.</li>
		<li>Mix the solutions without introducing air bubbles and pipette 70 &#956;L/spot onto the precoated comet slide. Using a pipette tip, spread the cell agarose mixture to form a thin layer. Place the slide in 4 &#176;C for 15 min.</li>
		<li>In a container, completely submerge the slide in cold lysis buffer. Place the container in the dark at 4 &#176;C for 1 h.</li>
		<li>Replace the lysis buffer with cold alkaline solution. Make sure the slides are completely submerged. Place the container in the dark at 4 &#176;C for 30 min.</li>
	</ol>
	</li>
	<li>Electrophoresis
	<ol>
		<li>Place the slide in a horizontal electrophoresis tank. Fill the tank with cold alkaline electrophoresis buffer until the slides are completely submerged. Apply voltage at 1 V/cm for 15 to 30 min. 
		NOTE: Total voltage = distance between the electrodes x 1 V</li>
		<li>In a container, completely submerge the slide in cold DI H2O. After 2 min, remove the DI H2O and add fresh DI H2O. Repeat this step.</li>
		<li>Replace the DI H2O with cold 70% ethanol. Wait 5 min. Gently remove the slide, do not tilt it, and let it air-dry.</li>
		<li>Once dried, add 100 &#956;L of diluted DNA dye to each spot. Wait 15 min at room temperature. Using a FITC filter in an epifluorescence microscope, take images of 50 to 100 comets in total per sample.</li>
	</ol>
	</li>
	<li>Image analysis and calculation of the results
	<ol>
		<li>For scoring the comets, use the National Institutes of Health&#8217;s (NIH) ImageJ with comet assay plugin (download ImageJ here: https://imagej.nih.gov/ij/download.html; download the plugin here: https://www.med.unc.edu/microscopy/resources/imagej-plugins-and-macros/comet-assay/). 
		NOTE: The plugin comes with a PDF instruction which contains all details to perform the analysis. Additionally, screenshots of the comet analysis are shown in Figure 2A-C. Representative comet images of control and doxorubicin (Doxo)-treated iPS cells and the quantification of the comets are shown in Figure 3A-C.</li>
	</ol>
	</li>
</ol>
2. DNA Damage Response
<ol>
	<li>Sample and reagent preparation
	<ol>
		<li>For an iPS-CM cell culture, culture iPS-CM cells in basement-membrane-matrix-coated (see Table of Materials) culture plates in RPMI medium supplemented with B-27 (CMM) until confluency is reached. 
		NOTE: Human-iPS-cell-derived cardiomyocytes were used in this protocol. The protocol for iPS cell differentiation into cardiomyocytes can be found in recent articles from our research group12,13,21.</li>
		<li>Seed iPS-CMs in chamber slides.
		<ol>
			<li>Discard the culture medium from the iPS-CM wells. Wash the cells three times with PBS.</li>
			<li>Add 1 mL of 0.25% trypsin per well and incubate at 37 &#176;C for 5 min. Check the plate under a microscope for cell detachment. Add 1 mL of CMM to stop the trypsin.</li>
			<li>Place cell suspension in a 15 mL tube and centrifuge for 5 min at 4 &#176;C and 200 x g. Discard the medium, add 1 mL of CMM, and resuspend the cells. Count the cells and adjust the cell count to obtain 20 x 105 cells in 1.6 mL of CMM.</li>
			<li>Seed 200 &#181;L of cell suspension per well of the 8-well chamber slide (see Table of Materials). Tap the slide gently to spread the cells around the well and incubate at 37 &#176;C.</li>
		</ol>
		</li>
		<li>For the iPS-CM doxorubicin treatment, discard the culture medium from the iPS-CM wells. Wash the cells with PBS. Treat the cells with 1 &#956;M doxorubicin and CMM for 4 h. Aspirate the medium and wash the cells with PBS.</li>
		<li>For the blocking buffer, prepare 10 mL of bovine serum albumin (BSA) solution containing 3% BSA and 0.05% Triton X-100. To prepare 10 mL of blocking buffer, add 1 mL of normal donkey serum to 9 mL of prepared BSA solution.</li>
		<li>For the primary antibody solution, add 1 &#956;L of anti-rabbit phospho-histone H2A.X (Ser139) antibody to 200 &#956;L of blocking buffer.</li>
		<li>For the secondary antibody mix, add 1 &#956;L each of fluorochrome-conjugated anti-rabbit secondary antibody, DAPI, and fluorochrome-conjugated phalloidin to 200 &#956;L of blocking buffer.</li>
	</ol>
	</li>
	<li>Immunolabeling
	<ol>
		<li>Wash the cells three times with PBS and add 200 &#956;L of freshly prepared 4% paraformaldehyde (PFA) to each well. Fix the cells for 20 min in the dark at room temperature. Wash the cells with PBS.</li>
		<li>Remove the PBS and add 200 &#956;L of blocking buffer per well and block for 30 min in a humidified chamber at room temperature. Remove the blocking buffer and add 200 &#956;L of primary antibody solution. Incubate for 45 min in a dark, humidified chamber at room temperature. Then, wash the wells three times with PBS.</li>
		<li>Remove the PBS and add 200 &#956;L of secondary antibody mix. Incubate for 45 min in a dark, humidified chamber at room temperature. Then, wash the wells three times with PBS. Wash with DI H2O before mounting them with antifade. Place a coverglass and store the slides in the dark at 4 &#176;C.</li>
		<li>Using an epifluorescence microscope, take three to five images of random fields per sample, as shown in Figure 4A, and proceed to image analysis.</li>
	</ol>
	</li>
	<li>Counting of the DDR foci
	<ol>
		<li>Download and install CellProfiler24 (http://cellprofiler.org).</li>
		<li>NOTE: Image processing in this study was performed using CellProfiler version 2.1.1. Tutorials for CellProfiler that were used can be found elsewhere (https://www.youtube.com/watch?v=PvhRL9xpduk&#38;list=PL7CC87670239B4D10). If a newer version of CellProfiler is being used, the pipeline might require minor adaptations.</li>
		<li>Download the pipeline punctae_gH2AX.cpipe. Select File &#62; Import &#62; Pipeline from the file and choose punctae_gH2AX.cpipe.</li>
		<li>Drag-and-drop the folder containing the acquired images of &#947;H2A.X foci to the File list window in the Input modules/Images section for analysis (Figure 5A). Then, follow the instructions as shown in Figure 5A-L.</li>
		<li>Set the following parameters for the images:
		<ol>
			<li>Drag the desired image for analysis onto the File list. Under Input modules, select Images (see Figure 5A for module settings). In Input modules, select Metadata (see Figure 5B for module settings). In Input modules, select NamesAndTypes (see Figure 5C for module settings).</li>
			<li>For Groups, select no. In Analysis modules, select ColorToGray (see Figure 5D for module settings).</li>
		</ol>
		</li>
		<li>In Analysis modules, select Smooth (see Figure 5E for module settings). 
		NOTE: This value may need to be optimized, depending on the image resolution.</li>
		<li>In Analysis modules, select IdentifyPrimaryObjects (see Figure 5F for module settings). 
		NOTE: Optimize these values to make sure the whole nucleus is selected. After this step, the computer may lag.</li>
		<li>In Analysis modules, select Smooth (see Figure 5G for module settings). 
		NOTE: This value may need to be optimized, depending on the image resolution.</li>
		<li>In Analysis modules, select EnhanceOrSuppressFeatures (see Figure 5H for module settings).</li>
		<li>In Analysis modules, select IdentifyPrimaryObjects (see Figure 5I for module settings). 
		NOTE: Optimize these values to make sure the punctae are selected. After this step, the computer may lag again.
		<ol>
			<li>In Analysis modules, select RelateObjects (see Figure 5J for module settings). In Analysis modules, select ClassifyObjects (see Figure 5K for module settings). In Analysis modules, select ExportToSpreadsheet (see Figure 5L for module settings).</li>
			<li>Click Analyze Images at the bottom left panel to perform image analysis. At the successful completion of the analysis, several images are generated (Figure 6A-F). 
			NOTE: Users should save these images for future reference.</li>
			<li>Note the .csv file containing the quantitative data for further analysis that is generated and saved in the appropriate location on the computer. In the spreadsheet called MyExpt_Image, columns B and D will have the number of nuclei that have up to five and more punctae, respectively. Add columns B and D to get the total number of nuclei.</li>
		</ol>
		</li>
		<li>Repeat step 2.3.3 - 2.3.8 for all images (change the MyExpt_ filename before every analysis). Plots can be made for assessing DNA integrity, as shown in Figure 4C.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

DNA integrity portrays cell integrity. Cells with damaged DNA are frequently in stress and eventually lose their integrity. The integrity of stem and stem-cell-derived cells that are being propagated for the purpose of transplantation is principal for the cells to perform their desired function. Transplanting cells with damaged DNA would result in a poor engraftment rate and performance of the cell8,20. Therefore, examining the DNA integrity prior to cell transplantation is a necessary quality control protocol. Here we describe two cost-effective approaches, namely the comet assay and &#947;H2A.X immunofluorescence labeling, to assess the DNA damage and quality of stem and stem-cell-derived cells.
A major cause of cellular aging is thought to be the accumulation of DNA damage in cells. Using the comet assay, Al-Baker et al. analyzed the DNA damage in young and senescent human dermal fibroblasts22. Previously, we have shown that transplanting DNA damage-free cardiac progenitor cells isolated from a p53 transgenic mouse increased the rate of engraftment in the host organ8. Recently, we have shown the role of the p53 transactivation domain in DNA damage repair mechanisms in human iPS cells21. In both studies, we have employed both the comet assay and &#947;H2A.X immunofluorescence labeling methodologies to assess the DNA damage in stem cells. Both these methods are cost-effective and can be performed with basic lab equipment. An annoying critical problem that we often experienced is agarose solidifying in the pipette and tubes. We overcome this issue by prewarming all the tubes and tips prior to pipetting agarose. While the comet assay takes up to 2 days to complete, the optimized &#947;H2A.X immunofluorescence labeling procedure can be completed in under 4 h.
Human iPS cell cultures with more than 10% DNA damage, measured by the comet assay, did not efficiently differentiate into cardiomyocytes (data not shown) in vitro. The comet assay is sensitive and dynamic in assessing DNA damage in stem cells. However, DNA integrity assessments in certain cells, such as iPS-derived cardiomyocytes, are cumbersome by comet assay due to the nature of the cells. The cardiomyocytes are enriched with troponin, sarcomeric proteins, and the secreted extracellular matrix proteins. Denaturing these complex structures to make supercoiled DNA and its reproducibility are questionable. Most importantly, 25% to 40% of human cardiomyocytes are binucleated25, and these percentages vary in iPS-derived cardiomyocytes. Since the cell wall is disrupted in the comet assay, the precise assessment of the percentage of DNA-damaged cells is impossible. Therefore, in the case of iPS-CMs, the &#947;H2A.X immunofluorescence labeling procedure to assess DNA damage is a simplistic alternative for the comet assay.
Based on these results and those from previous publications by our group8,20,21, we suggest that, if there is more than 10% DNA damage, assessed by the comet assay, or less than 90% of the cells have zero to five DDR foci, then the culture should be disqualified for cell transplantation.

Experimental and clinical evidence demonstrates that cell transplantation can moderately improve the left ventricle contractile performance of failing hearts1,2,3,4,5,6,7,8,9. Recent advances have opened further appealing opportunities for cardiovascular regeneration; these include the forced expression of reprogramming factors in somatic cells to induce pluripotency and differentiate these induced pluripotent stem cells (iPSC) into different cardiac lineages, importantly cardiomyocyte (CM)10,11,12,13. The hereditary material in every cell, including the artificially generated iPSCs and iPSC-derived CM (iPS-CM), is DNA. The genetic instructions stored in DNA dictates the growth, development, and function of cells, tissues, organs, and organisms. DNA is not inert; cell metabolites, such as reactive oxygen, carbonyl, and nitrogen species, and alkylating agents can cause DNA damage in vitro and in vivo14,15,16,17. Importantly, DNA damage occurs intuitively in every cell, with a significant frequency. If these damages are not corrected, it will lead to DNA mutation, cellular senescence, the loss of DNA and cell integrity, and possibly, diseases, including life-threatening cancers. Therefore, retaining DNA integrity is essential to any cell, especially iPSCs, that has enormous potential in the clinic.
For assessing the quantity and integrity of isolated genomic DNA, expensive equipment is available on the market. However, there are no simple and cost-effective methods to assess the DNA integrity in cells without isolating the cells. Moreover, user-induced DNA degradation during DNA isolation is one of the major drawbacks in using these methods. The single-cell gel electrophoresis (known as comet assay)18,19 and &#947;H2A.X immunolabeling8 techniques are fundamental approaches in research labs for assessing DNA damage. These two methods do not require expensive equipment or isolated genomic DNA to analyze DNA integrity8,20,21. Since, these techniques have been performed with whole cells; user-induced DNA/RNA/protein degradation during the sample preparation will not affect these protocols. Here, to assess the DNA damage and DNA damage response in stem and stem-cell-derived cells, we provide step-by-step protocols to perform both the comet assay and &#947;H2A.X immunolabeling. Moreover, combining these two approaches, we propose a naive assessment that can be used to evaluate the cell&#39;s suitability for transplantation.
The comet assay, or single-cell gel electrophoresis, measures the DNA breaks in cells. Cells embedded in low-melting agarose are lysed to form nucleoids containing supercoiled DNA. Upon electrophoresis, small pieces of fragmented DNA and broken DNA strands migrate through the agarose pores, whereas the intact DNA, due to their enormous size and their conjugation with the matrix protein, will have a restricted migration. The pattern of stained DNA under a fluorescence microscope mimics a comet. The comet head contains intact DNA and the tail is composed of fragments and broken DNA strands. The fraction of DNA damage can be measured by the fluorescence intensity of the damaged DNA (comet tail) relative to the intact DNA (comet head) intensity. The parameter tail moment can be calculated as shown in Figure 1.
DNA damage induces the phosphorylation of histone H2A.X (&#947;H2A.X) at Ser139 by ATM, ATR, and DNA-PK kinases. The phosphorylation and recruitment of H2A.X at DNA strand breaks is called DNA damage response (DDR) and happens rapidly after DNA is damaged. Following this process, checkpoint-mediated cell cycle arrest and DNA repair processes are initiated. After the successful completion of DNA repair, &#947;H2A.X is dephosphorylated and inactivated by phosphatases. Prolonged and multiple DNA strand breaks lead to the accumulation of &#947;H2A.X foci in DNA. This indicates the cell&#39;s inability to repair the DNA damage and the loss of DNA integrity. These &#947;H2A.X foci in DNA can be identified by and the number of DDR foci can be counted using the protocol in section 2.

<table><tbody><tr><td>0.25% Trypsin</td><td>Corning</td><td>25200056</td><td></td></tr><tr><td>8-well chamber slide</td><td>Millicell</td><td>PEZGS0816</td><td></td></tr><tr><td>Accutase</td><td>Stemcell Technologies</td><td>7920</td><td>Cell detachment solution</td></tr><tr><td>Alexa Fluor 488 AffiniPure&lt;br/&gt; Donkey Anti-Rabbit IgG</td><td>Jackson Immuno&lt;br/&gt; Research Laboratories</td><td>711-545-152&lt;br/&gt; (RRID: AB_2313584)</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Cy3 AffiniPure Donkey Anti-Rabbit IgG</td><td>Jackson Immuno&lt;br/&gt; Research Laboratories</td><td>711-165-152&lt;br/&gt; (RRID:AB_2307443)</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9564</td><td></td></tr><tr><td>Gibco B-27 Supplement, Serum Free,</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>Matrigel growth factor reduced</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>mTeSR1</td><td>Stemcell Technologies</td><td>85850</td><td></td></tr><tr><td>PhalloidinA488</td><td>Life Technology</td><td>A12379</td><td></td></tr><tr><td>Phospho-Histone H2A.X (Ser139) Antibody</td><td>Cell Signaling Technology</td><td>2577 (RRID: AB_2118010)</td><td></td></tr><tr><td>RPMI</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>SYBR&amp;nbsp;Green I nucleic acid gel stain</td><td>Sigma-Aldrich</td><td>S9430</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher scientific</td><td>BP151-100</td><td></td></tr><tr><td>UltraPure Low melting Point Agarose</td><td>Invitrogen</td><td>16520050</td><td></td></tr><tr><td>Vectashield</td><td>Vector Laboratories</td><td>H-1000</td><td>Antifade</td></tr></tbody></table>

assessing stem cell dna integrity for cardiac cell therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Human induced pluripotent stem cells were cultured, and the DNA damage and the tail moment, which were used as a measure of DNA integrity, were analyzed by comet assay. iPS cells were embedded in low-melting-point agarose and placed on a glass slide. The cells were, then, treated with lysis buffer, followed by an alkaline solution, to obtain supercoiled DNA. Nucleoids were electrophoresed and comets were visualized by DNA dye (Figure 1A-D). The comets were, then, analyzed with ImageJ, using the comet assay plugin (Figure 2A-C).
Human iPS cells were treated with Doxo to induce DNA damage and to be used as a positive control. Representative micrographs of comets from Doxo-treated and nontreated iPS cells are shown in Figure 3A. A basal amount of DNA damage was found in iPS cells, expressed as a fraction of DNA damage and tail moment. However, the Doxo treatment increased the DNA damage in iPS cells as expected (Figure 3B,C). This shows that the comet assay can be used to assess DNA integrity not only in somatic cells18,19 but also in pluripotent stem cells21.
Freshly differentiated iPS cardiomyocytes (iPS-CMs), iPS-CMs cultured for 6 months (prolonged culture [PC]), and iPS-CMs treated with Doxo were subjected to &#947;H2A.X immunolabeling. Representative micrographs of &#947;H2A.X immunolabelling are shown in Figure 4A (lower-magnification) and Figure 4B (higher-magnification). The number of &#947;H2A.X foci (DDR foci) (punctae) in each nucleus are quantified, using CellProfiler with custom pipeline modules (Figure 5A-L). The percentage of cells that are positive for &#947;H2A.X are classified into nuclei with zero to five punctae and nuclei with more than 10 punctae. In the control iPS-CM, more than 90% of the cells had less than five DDR foci per nuclei, and a total of less than 10% of the cells had more than six DDR foci per nuclei (Figure 4C, control [Ctrl]). iPS-CMs cultured for 6 months had less than 90% cells with less than five DDR foci per nuclei, and a total of more than 13% of the cells had more than six DDR foci per nuclei (Figure 4C, PC), whereas the Doxo-treated iPS-CM showed less than 80% of the cells with less than five DDR foci per nuclei, and a total of about 24% of the cells had more than six DDR foci per nuclei (Figure 4C, Doxo). This data clearly shows that prolonged cell culture and Doxo treatment induce significant DNA damage in iPS-CMs and are not suitable for cell transplantation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58971/58971fig1.jpg" /> 
Figure 1: Schematic of the comet assay. (A) Mix the cell suspension with low-melting-point agarose and (B) place it on a glass slide. (C) Treat it with cell lysis buffer, followed by an alkaline solution, to get nucleoids containing supercoiled DNA. (D) Electrophorese and stain the DNA using SYBR green DNA dye. (E) Schematic of intact (left) and damaged DNA (right, comet shape). (F) Formula to calculate a fraction of the DNA damage and tail moment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58971/58971fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58971/58971fig2.jpg" /> 
Figure 2: DNA damage and tail moment quantification by ImageJ. (A-C) Screenshots of ImageJ, with the comet analysis plugin showing a selection of the comet head and tail. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58971/58971fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58971/58971fig3.jpg" /> 
Figure 3: Doxorubicin induces DNA damage in human iPS cells. (A) DNA damage in doxorubicin-treated (Doxo) and nontreated (control) iPS cells, analyzed using the comet assay. (B) Fraction of DNA damage (n = 3) and (C) tail moment (n = 63 comets) quantified using the comet assay. Treatment with doxorubicin, a DNA-intercalating agent, significantly increased the fraction of DNA damage and tail moment in iPS cells. Data are means &#177; SEM. *P &#60; 0.05, Student&#39;s unpaired t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58971/58971fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58971/58971fig4.jpg" /> 
Figure 4: DNA damage response in iPS-derived cardiomyocytes. (A) DNA damage response marker &#947;H2A.X identified by immunofluorescence in doxorubicin-treated (Doxo) and nontreated (control) iPS-cardiomyocytes as well as prolonged culture iPS-cardiomyocytes (PC). (B) Higher magnification of &#947;H2A.X (green punctae) labeling at the sites of DNA damage in iPS-CMs. (C) Quantification of DDR foci, analyzed using CellProfiler with custom pipeline modules. Data are means &#177; SD; n = 3 to 4. *P &#60; 0.05, Student&#39;s unpaired t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58971/58971fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58971/58971fig5.jpg" /> 
Figure 5: Automated DNA damage response analysis by CellProfiler. (A-L) Screenshots of CellProfiler with specified settings for importing the images, and the automated identification and quantification of &#947;H2A.X punctae in iPS-CMs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58971/58971fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58971/58971fig6.jpg" /> 
Figure 6: Automated DNA damage response analysis by CellProfiler. (A-F) Data images generated by CellProfiler at the completion of the image analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58971/58971fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy at JoVE.com

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

assessing-stem-cell-dna-integrity-for-cardiac-cell-therapy

Nanyang Technological University

Research

JoVE Journal

Genetics

7.6K Views.  University of Alabama at Birmingham. Voor neonatale ziekten zoals cardiomyopathie, regeneratieve therapie met behulp van geïnduceerde pluripotente stamcel-afgeleide cellen heeft een enorm potentieel. Echter, in vitro versnelde celgroei leidt tot DNA-schade door geaccumuleerde metabolieten zoals reactieve zuurstofsoorten. Het transplanteren van deze DNA-beschadigde cellen zou leiden tot een slechte engraftment en regeneratie van het orgaan.Vóór transplantatie moet de kwaliteit van de cellen worden beoordeeld. Hier biede...

Video: Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4&#8242;,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

Developmental Biology

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Medicine

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Biochemistry

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Immunology and Infection

Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Biology

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.

The present protocol describes all-optical control and observation of triggered cellular activity in iPSC-derived cardiomyocytes (iPSC-CMs) for high throughput drug screening and toxicity testing. Multi-parametric quantification of phenotypic patterns in time, and space, are shown. Long-term effects of drugs over hours, or sequential measurements over days, are demonstrated.

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. ...

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

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